Technical Field
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The present invention relates to, in a multi-capillary
electrophoretic apparatus comprising such a multi-capillary array
migration part that a plurality of capillary columns are arranged so that
samples are injected into the respective capillary columns and
simultaneously electrophoresed in all capillary columns and an optical
measuring part irradiating capillaries with light in the multi-capillary array
migration part and measuring absorbance by the samples in the irradiated
parts and fluorescence from the samples, a capillary cassette forming
the multi-capillary array migration part.
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Such a multi-capillary electrophoretic apparatus is used for
separating/analyzing nucleic acid, protein, peptide, sugar and the like, and
particularly plays an important role for analysis of the base sequence of
DNA. The multi-capillary electrophoretic apparatus for sequence
determination of DNA employs Sanger's reaction, electrophoreses a DNA
fragment (fragment) sample labeling a primer or a terminator with a
fluorescent material and detects fluorescence from the DNA fragment
sample during migration for deciding the base sequence.
Background Technique
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For sequence determination of DNA having long base sequence
such as a human genome, a DNA sequencer having high sensitivity, a high
speed and large throughput is necessary. As one method thereof, a
multi-capillary DNA sequencer arranging capillary columns charged with
gels in plural is proposed in place of that employing flat plate type slab
gels. In the capillary column, a sample is not only easy to handle or
inject but also can be migrated at a high speed and detected in high
sensitivity as compared with the slab gel. Namely, such problems result
from influence by Joulean heat that a band spreads and a temperature
gradient takes place applying a high voltage in the slab gel, while such
problems are less in the capillary column and spreading of a band is small
to allow high sensitivity detection even if making high-speed migration
while applying a high voltage.
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When making electrophoresis employing a plurality of capillary
columns, it is desirable that attachment/detachment of the capillary
columns to/from an electrophoretic apparatus body is easy, and it is
desirable to cassette the same with a holder for that In consideration
of the recent environmental problem, it is also preferable that the
capillary columns can be readily separated from the holder when
discarded after use.
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Furthermore, since a step by pressurization or decompression is
requisite for charging gels into the capillary columns, airtightness is
necessary for fixation of the capillary columns to the holder at least on
sample injection sides in order to simultaneously charge the gels into all
capillary columns contained in the cassette.
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Accordingly, a first objective of the present invention is to
provide a capillary cassette which can readily separate capillary columns
from a holder and has sufficient airtightness between the capillary
columns and the holder on sample injection sides.
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The capillary columns are so thin that the same are difficult to
manually work and are easily breakable. When fixing the capillary
columns to the holder, it is a difficult operation requiring skill to bundle
the same in plural, and in addition, it requires a long time.
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Accordingly, a second objective of the present invention is to
make it possible to simply and quickly perform preparation of a capillary
cassette.
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The capillary columns are protected with various coats, in order
to increase mechanical strength. For coats, polyimide, silicone resin,
polytetrafluoroethylene, acrylic resin and the like are used. When using
the same as coats for capillary columns of electrophoresis however,
optical characteristics of the coats such as light absorption and
fluorescence hinder detection since optical means for absorbance
measurement and fluorescence measurement are employed for
detection of samples migrating in the capillary columns. Therefore,
when the coats are made of materials hindering optical detection, the
coats of detection parts are burned by flames and removed with burning
means such as a lighter or a gas burner.
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A multi-capillary electrophoretic apparatus using a plurality of
capillary columns and simultaneously detecting a plurality of samples
aligns, arranges and fixes such capillary columns that coats of detection
parts are removed, thereby forming detection windows when the coats
are made of materials hindering optical detection.
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While a number of capillary columns are arranged in the multi-capillary
electrophoretic apparatus, it follows that when performing coat
removal of the detection parts on every one of the capillary columns, a
long time is required for the operation.
-
Furthermore, the coating materials include such a one that a part
located in a flame is completely burned and removed in the process of
removing the coating material by burning by the flame while the same is
melted but does not come to be removed in a portion separate from the
flame and convex parts resulting from solidification of the melted coat
are formed on both sides of the part from which the coat is removed. If
such convex portions are formed, adjacent capillary columns do not
adhere to each other or come to different levels due to the convex
portions and detection windows are not planarly aligned when arranging
the capillary columns, aligning positions of the parts from which the coats
are removed and forming the detection windows. Consequently, the
focus of an optical system may so deviate in detection that it cannot
perform proper detection.
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Accordingly, a third objective of the present invention is, when a
coat for a capillary column is made of a material hindering optical
detection, to provide such a capillary cassette that a detection window of
a capillary column employed for a multi-capillary electrophoretic
apparatus is planarly formed to be suitable for optical measurement and a
method of manufacturing a capillary cassette having a feature in a
detection window forming step thereof.
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An initial capillary electrophoretic apparatus is that employing a
single capillary column. In this case, it dips an end of the capillary
column into a gel solution in a vessel, closes the vessel and pressurizes
the vessel thereby pushing the gel into the capillary column and charging
the same as a method of charging the gel into the capillary column. As
another method, an operation of dipping an end of the capillary column
into the gel solution, decompressing another end of the capillary column
and sucking the gel into the capillary column, thereby charging the same,
is also performed.
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In multi-capillary electrophoresis, it is necessary to align capillary
column end surfaces on sample injection sides in sample injection while it
is necessary to align and handle capillary coat removing parts on
detection sides when performing detection for handling a plurality of
capillary columns, and hence it is desirable to provide such a capillary
cassette that arrangement is fixed by a holder so that both the sample
injection sides and the detection sides are in prescribed arrangement
relation.
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Furthermore, it is extremely troublesome to charge a gel into
every one of the capillary columns in the state of the capillary cassette,
and, in actuality, impossible in practice. Therefore, awaited is means for
making it possible to simply perform gel charging into all capillary columns
included therein in units of the capillary cassette.
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Accordingly, a fourth objective of the present invention is to
provide an apparatus to make it possible to simply charge gels into all
capillary columns included in such a capillary cassette.
Disclosure of the Invention
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In order to attain the first objective, the present invention is such
that in a capillary cassette two-dimensionally arranging and fixing a
plurality of capillary columns used in a multi-capillary electrophoretic
apparatus on sample injection sides by a holder while arranging the same
in a line on a plane on detection sides (sides opposite to the sample
injection sides), the holder comprises a rubber plate so that end portions
of the plurality of capillary columns pass through the rubber plate one by
one to be fixed by elastic force of the rubber plate.
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By inserting the capillary columns into the rubber plate and fixing
the same with the holder on the sample injection sides, airtightness
between the rubber plate and the capillary columns can be maintained
and it is possible to serve a sealing function with respect to
pressurization or suction in gel charging into capillaries. The capillary
columns are not fixed by an adhesive or the like, whereby the capillary
columns can be readily extracted from the holder in disposal of the
capillary columns or the like.
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In the present invention, it is preferable to comprise another
holder pressing and holding the capillary columns from at least single
surface sides of the plurality of capillary columns arranged in a line on the
plane through the rubber plate on the detection sides of the capillary
columns. By thus making fixation on the detection sides by pushing the
capillary columns through the rubber plate, it is easy to detach the
capillary columns from the holder on the detection sides, and an
operation of separating the capillary columns and the holder in disposal
after use or exchanging defective capillary columns becomes easy.
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Furthermore, the holder on the detection sides preferably
comprises clamping means fixing the capillary columns on both side
portions while pressing and fixing the same on at least two portions of an
intermediate part as means fixing the plurality of capillary columns
arranged in a line on the plane through the rubber plate. By making it
possible to press the capillary columns also in at least two portions of the
intermediate part, it becomes easy to maintain arrangement on the plane.
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A capillary cassette preparation method according to the present
invention to attain the second objective is a method of passing through a
supporter of a prescribed fixing position of capillary column fixing means
having the supporter consisting of a rubber plate with a needle having an
inner diameter larger than a capillary column from its vertical direction,
guiding the capillary column into the needle and making the same pass
through the supporter, thereafter extracting only the needle thereby
holding and fixing the capillary column by the supporter and repeating this
operation while changing the fixing position on the supporter thereby
successively two-dimensionally arranging and fixing a plurality of capillary
columns.
-
The capillary column guided in the needle to pass through the
supporter of the rubber plate on a sample injection side holder (capillary
column fixing means) is held and fixed by elastic force of rubber after
only the needle is extracted from the supporter.
-
A capillary cassette preparation apparatus according to the
present invention for executing this capillary cassette preparation
method comprises arrangement position decision means holding capillary
column fixing means having a supporter consisting of a rubber plate for
moving and fixing the same in an in-plane direction of the supporter, a
needle passing through the supporter comprised in the capillary column
fixing means from a vertical direction of its plane and guiding a capillary
column to the fixing position, a first capillary column holder holding the
capillary column and inserting the same into the needle, a guide guiding
the forward end of the capillary column held by the first capillary column
into the needle, slide guide means moving the needle, the guide and the
first capillary column holder in a rectilinear direction perpendicular to the
plane of the supporter, a second capillary column holder holding the
forward end portion of the capillary column made to project from the
forward end of the needle in such a state that the needle passes through
the supporter of the capillary column fixing means, cut means cutting the
capillary column in a prescribed length, and a roller unit successively
feeding the capillary column in an insert direction.
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The fixing position of the capillary column is decided by mounting
the capillary column fixing means on the arrangement position decision
means and moving the arrangement position decision means upward,
downward, leftward and rightward. It linearly moves the needle in the
capillary column fixing means direction along the slide guide means and
makes the same pass through the supporter on the fixing position. The
forward end of the capillary column wound on a capillary drum is guided
into the needle by the guide following the needle along the slide guide
means and the first capillary column holder, and projects from the
forward end of the needle. It holds the forward end portion of the
capillary column by the second capillary column holder comprised on a
needle projection side of the arrangement position decision means, and
extracts only the needle from the supporter of the capillary column fixing
means. The capillary column forward end portion remains in the
supporter, elastic force of rubber which is the supporter acts in a
direction closing a hole opened by the needle, and the capillary column is
fixed to the capillary column fixing means. It cuts the fixed capillary
column in the prescribed length by the cut means. The arrangement
position decision means moves and brings a next fixing position onto a
moving straight line of the needle, the guide and the capillary column
holder. The capillary column wound on the drum is timely fed by the
roller unit having a motor for motive power.
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According to this capillary cassette preparation apparatus,
capillary columns of a predetermined length are aligned in determined
order by operating a lever in accordance with a prescribed procedure so
that a capillary cassette can be prepared, whereby the efficiency of a
preparation operation for the capillary cassette remarkably rises.
Furthermore, it can simply make transition to automatization by mounting
a driving source, whereby simple preparation of a capillary cassette with
less failure is enabled.
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In order to also form detection windows simultaneously with
capillary cassette preparation, it is preferable to further comprise
detection window preparation means removing capillary column coats of
prescribed positions from the forward ends of the capillary columns and
preparing the detection windows and arrangement means arranging
terminating end sides of the cut capillary columns in a line in determined
order in the capillary cassette preparation apparatus.
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After the capillary columns are guided in the needle and pass
through the capillary column fixing means, a voltage is applied to the
detection window preparation means to prepare the detection windows
on prescribed positions from the forward ends of the capillary columns.
Thereafter the terminating ends of the capillary columns cut in the
prescribed length are successively dropped between planes and arranged
in a line by the arrangement means formed by two planes having a parallel
clearance slightly wider than the capillary column outer diameter.
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A capillary cassette according to the present invention for
attaining the third object is such a one that a plurality of capillary
columns are fixed by a holder on sample injection sides and planarly
arranged in a line on detection sides, coats of the capillary columns are
removed so that strip detection windows extending in the capillary
column arrangement direction are formed on the capillary column
arrangement on the detection sides, the capillary columns adhere to
each other and the coats fuse to each other so that the capillary column
arrangement is integrated around the detection windows.
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In this capillary cassette, the capillary columns adhere to each
other, the coats fuse to each other and the capillary column arrangement
is integrated around the detection windows, whereby the detection
windows planarly align, the focus of an optical system in detection does
not deviate, and it is possible to perform correct detection.
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When manufacturing the capillary cassette, a manufacturing
method according to the present invention forms the detection windows
including the following steps (A) and (B):
- (A) a step of fixing the sample injection sides of the capillary
columns using the holder and thereafter arranging and holding the
capillary columns in a line planarly in close contact with each other, and
- (B) a step of bringing heating means having a length for a plurality
of capillary column outer diameters into contact with or approximating
the same to a detection window formation planned region of the held
capillary column arrangement for removing coats of the plurality of
capillary columns while melting the coats around regions from which the
coats are removed and thereafter solidifying the same for making the
coats of the adjacent capillary columns fuse to each other.
-
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It planarly arranges and fixes the capillary columns not subjected
to coat removal of detection parts in a line and simultaneously performs
coat removal of detection window formation planned positions of the
plurality of capillary columns with the heating means. By simultaneously
heating and removing the coats of the plurality of capillary columns, the
adjacent capillary columns are fused and integrated with each other on
positions separate from the regions from which the coats are removed
when the melted coats are cooled and solidified. Thus, the plurality of
capillary columns arranged in a line form a flat cable.
-
For a means of heating a nichrome wire heater or a ceramic
heater can be employed.
-
When using as the heating means employed for removing the
coats that whose length is shorter than the width of the capillary column
arrangement on the detection sides, it repeats the step (B) a number of
times in the width direction of the capillary column arrangement for
forming the detection windows.
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This manufacturing method planarly arranges the plurality of
columns and thereafter simultaneously removes the coats of the
detection parts of the plurality of capillary columns, whereby remarkable
reduction of the detection window preparation time can be made as
compared with the case of removing the coat as to every single capillary
collar.
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In the peripheral parts of the detection windows formed by this
manufacturing method, the coats melted but not come to be removed
fuse the adjacent capillary columns when cooled and solidified. The
plurality of fused capillary columns are previously planarly arranged and
hence come to planarly maintain the detection windows after fusion, and
it is possible to readily manufacture a capillary cassette capable of
performing correct optical detection.
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When fixing the detection sides of the plurality of capillary
columns, it saves trouble of fixing the same one by one while it does not
fix the same with an adhesive, whereby a time for drying the adhesive can
also be saved.
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A gel charging apparatus according to the present invention for
attaining the fourth object is an apparatus for charging gels into capillary
columns of a capillary cassette. In the capillary cassette, end portions
of sample injection sides of a plurality of capillary columns mounted on a
multi-capillary electrophoretic apparatus are two-dimensionally arranged
while passing through a holding member of a holder and fixed while
keeping airtightness between the same and the holding member.
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Gel charging into the capillary columns can be performed either
by suction or pressurization. In gel charging it does not seal the
respective capillary columns one by one to perform suction or
pressurization but seals all capillary columns of the capillary cassette by
the holder fixing the capillary columns while keeping airtightness to
simultaneously perform gel charging.
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In the system performing gel charging by suction, the present
invention comprises a chamber provided with an opening in its upper
surface so that closure means introducing end portions on sample
injection sides of the capillary columns inside and closing the opening
with the holder is provided in the opening and further provided with an
exhaust port and a release port to the atmosphere, a gel vessel storing a
gel solution so that end portions of the capillary columns opposite to the
sample injection sides are dipped therein, exhaust means provided on the
exhaust port of the chamber, and a switching valve provided on the
release port
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When performing gel charging by suction, it dips the forward ends
of the detection sides of all capillary columns of the capillary cassette in
gels contained in the gel vessel, closes the opening of the chamber with
the holder for the capillary cassette, decompresses the chamber with the
exhaust means and inhales the gel solution into the capillary columns.
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In the system performing gel charging by pressurization, the
present invention comprises a chamber provided with an opening on its
upper surface so that closure means introducing end portions of the
sample injection sides of the capillary columns inside and closing the
opening with the holder is provided in the opening while storing a gel
solution on such a position that the end portions on the sample injection
sides of the capillary columns are dipped therein in such a state that the
opening is closed with the holder and further provided with a
pressurization port and a release port to the atmosphere, pressurization
means provided on the pressurization port of the chamber and a
switching valve provided on the release port.
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When performing gel charging by pressurization, it introduces the
gel solution into the chamber, dips the forward ends of the capillary
columns on the sample injection sides in the gel solution, closes the
opening of the chamber with the holder, pressurizes the chamber with
the pressurization means and pressure-fits gels into the capillary
columns.
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When charging the gel solution into the plurality of capillary
columns forming a capillary array mounted on a multi-capillary
electrophoretic apparatus, it does not directly close/fix the respective
capillary columns but seals the plurality of capillary columns to the
closure means with the sample injection side holder fixing the same with
excellent airtightness to perform charging of the gels, whereby gel
charging for the plurality of capillary columns can be simultaneously
performed. Furthermore, simple mounting and airtightness in mounting
can be compatibly attained.
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Charging of the gel solution into the plurality of capillary columns
can be simultaneously performed, whereby it does not damage the
maximum merit of improvement of throughput by simultaneous
performance of migration of a plurality of samples in multi-capillary
electrophoresis in the process of gel preparation which is the
pretreatment thereof.
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It is preferable that a flow control valve controlling the flow rate is
provided between the release port of the chamber and the switching
valve. While the pressure in the chamber is released to the atmosphere
after gel charging into the capillary columns, the speed at which the
internal pressure of the chamber returns can be controlled with the flow
control valve. While air may be mixed into the gels charged into the
capillary columns when the degree of decompression or pressurization in
the chamber is large, the degree of decompression or pressurization can
be controlled with the flow control valve in this case.
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In the system performing gel charging by pressurization, the gel
solution may be directly introduced into the chamber, while it is
preferable in the aspect of maintenance of the chamber when providing
an attachable/detachable vessel in the chamber for storing the gel
solution in the vessel.
Brief Description of the Drawings
-
- Fig. 1 is a schematic perspective view showing an example of a
multi-capillary electrophoretic apparatus to which a capillary cassette
according to the present invention is applied. Fig. 2 is a front elevational
view of a capillary cassette of one embodiment Fig. 3 is a left side
elevational view of the capillary cassette of the embodiment. Fig. 4 is a
top plan view of the capillary cassette of the embodiment Fig. 5 shows
diagrams mounting a carrier on the embodiment, (A) is a front elevational
view, (B) is a left side elevational view, and (C) is a top plan view. Fig. 6
is a schematic perspective view of one embodiment of a capillary
cassette preparation apparatus according to the present invention. Fig.
7 is a plan view of the embodiment Fig. 8 is a front elevational view of
the embodiment Fig. 9 is a flow chart of the operation procedure of the
embodiment Fig. 10 is a schematic perspective view showing the final
step of one embodiment of a capillary cassette manufacturing method
according to the present invention. Fig. 11 is a front elevational view
showing a schematic structure of one embodiment of a capillary column
gel charging apparatus according to the present invention in a partially
fragmented manner. Fig. 12 is a plan view showing the embodiment in a
partially fragmented manner. Fig. 13 is a sectional view showing the
embodiment in a partially fragmented manner, and that showing a state
cut on an A-A line position of Fig. 12.
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Best Modes for carrying out the Invention
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Fig. 1 shows a schematic perspective view of an example of a
multi-capillary electrophoretic apparatus to which a multi-capillary
cassette according to the present invention is applied.
-
A capillary cassette 2 is such a one that a plurality of capillary
columns 102 are arranged and fixed by holders 4 and 6 to form a capillary
array, and an end 2a of the capillary array defines a sample injection side
and is two-dimensionally arranged and fixed by the holder 4, to come into
contact with a buffer solution of a reservoir 62 for migration after sample
injection. A terminating end 2b of the capillary array is such that the
capillary columns 102 are arranged in a line on a plane, to come into
contact with a buffer solution of a reservoir 56 on the forward end.
Such a detected portion 2c that the capillary columns 102 are arranged
in a line and supported by the holder 6 is provided on a terminating end
side (detection side) of the capillary array. The capillary columns 102
are coated with coats to be protected against breakage. When a
fluorescent detection method is employed for detecting migrating
samples and if the coats emit fluorescence, it removes the coats on the
detected portion 2c. When using those coated with coats which are
transparent and made of a non-fluorescent material, the coats of 102 of
the capillary columns do not have be removed also on the detected
portion 2c. A number of capillary columns, e.g., 384 capillary columns
are arranged on the capillary cassette 2.
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Different samples are injected into the respective capillary
columns 102, and electrophoresis is simultaneously performed.
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For an excitation light source 8 to be used for exciting the
samples themselves or fluorescent materials labeling the samples, an
argon gas laser unit, for example, is provided. 10 is an
excitation·photoreceiving optical system, which is that irradiating the
capillary columns 102 of the detected portion 2c with excitation light and
detecting fluorescence from the samples, and scanned by a scanning
mechanism (illustration omitted) in a scan direction parallel to the
arrangement plane of the capillary cassette in the detected portion 2c
and perpendicular to a migration direction. In order to make the
excitation light beam from the excitation light source 8 not deviate even
by scanning of the excitation·photoreceiving optical system 10, the laser
beam from the excitation light source 8 is guided to the
excitation·photoreceiving optical system 10 through optical fiber 9
coupled by a coupler here as an example.
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A migration buffer solution is contained in the lower reservoir 62,
the lower end 2a of the capillary array is dipped in the buffer solution, and
a migration voltage is applied to the capillary ends of the lower end 2a of
the capillary cassette 2 through the buffer solution. An upper electrode
54 is dipped in and comes into contact with the buffer solution of the
upper reservoir 56, a lower electrode 58 is dipped in and comes into
contact with the buffer solution of the lower reservoir 62, and the
migration voltage is applied to both electrodes 54 and 58 from a high
pressure power source 60. Its power supply voltage is 30 kV, for
example, and a current capacity is 10 to 30 mA.
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The migrated samples are protein samples, DNA fragments
labeled with a fluorescent material or the like.
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Fig. 2, Fig. 3 and Fig. 4 show schematic block diagrams of a
capillary cassette of one embodiment Fig. 2 is a front elevational view,
Fig. 3 is a left side elevational view, and Fig. 4 is a top plan view. The
same reference numerals are assigned to parts playing the same roles as
Fig.1.
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A holder 4 on a sample injection side has a coat and is such a
one that a rubber plate 14 made of silicone rubber of 5 mm in thickness
holding and fixing capillary columns 102 with a hole is held between
holder plates 4a and 4b of resin for two-dimensionally arranging the
quartz glass capillary columns 102 of 300 µm in outer diameter and 100
µm in inner diameter, for example, and integrated by fixing screws 4c.
384 holes passing the capillary columns 102 therethrough are provided
on both holder plates 4a and 4b in two dimensions of 16 by 24 of 4.5 mm
pitches in correspondence to positions of respective holes of a 384-hole
microplate used for sample introduction. The holes of the holder plates
4a and 4b are set to be larger than the outer diameters of the capillary
columns 102. The capillary columns 102 pass through both holder
plates 4a and 4b and the rubber plate 14 held therebetween and are held
in the holes of the rubber plate 14 by elasticity of rubber, thereby being
fixed while keeping airtightness between the same and the holder 4. A
carrier fixture 20 is provided on an end of the holder plate 4a.
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A holder 6 on a detection side fixes the capillary columns 102
closely arranged on a plane by holding the same with a holder plate 6a
from below and with a rubber plate 16 of silicone of 2 mm in thickness
from above. In order to press the capillary columns 102 against the
holder 6 with the rubber plate 16 and fix the same, holder plates 6b fixing
the rubber plate 16 to the holder plate 6a by fixing screws 6c are
provided on both side portions of the arrangement of the capillary
columns 102.
-
When arranging the 384 capillary columns 102 in a line in the
holder 6, the width becomes about 12 cm even if arranging the same in
close contact and hence the rubber plate 16 is bent outward at the
central portion when fixing both end portions, and hence the force of the
rubber plate 16 holding the capillary columns 102 weakens around the
center of the holder 6. So that the rubber plate 16 does not float in a
direction separating from the holder plate 6a between both end portions
at which the rubber plate 16 is fixed to the holder plate 6a by the holder
plate 6b, two grooves 17a extending in the arrangement direction of the
capillary columns 102 are provided inside (the side opposed to the rubber
plate 16) of the holder plate 6b, clamp bars 18b pushing the rubber plate
16 in the direction of the holder plate 6a are engaged in several portions,
e.g, in positions of six portions in total including three portions on either
side, and a clamp screw 18a fitted with the holder plate 6b to project
outward from the holder plate 6b is provided in each clamp bar 18b. By
adjusting the clamp screw 18a, clamping force pressing the capillary
columns 102 through the rubber plate 16 can be adjusted.
-
The total length of the capillary columns 102 is about 500 mm,
and the detected portion 2c is provided on a position of about 400 mm
from a sample injection end. In order to form detection windows on the
detected portion 2c, a slot 17c along the arrangement direction of the
capillary columns 102 is opened in the holder plates 6a and 6b and the
rubber plate 16 to define the detected portion 2c. Signal detection in
electrophoresis is performed through the slot 17c.
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In order to fix the capillary columns 102 to the holder 4, it holds
the rubber plate 16 between the holder plates 4a and 4b, passes the
rubber plate 14 with a needle along the opening holes of the holder plate
4b, thereafter inserts the capillary columns 102 into the needle, and
extracts only the needle, as described later with reference to Fig. 6 to Fig.
9 in detail. Thus, the capillary columns 102 pass through the rubber
plate 14, and are supported and fixed by the rubber plate 14 in a state
held by elastic force of rubber. Thereafter it cuts the capillary columns
102 in a prescribed length. By repeating this operation while changing
the positions of the holes of the holder plate 4b, the capillary columns 2
are two-dimensionally arranged and fixed to the holder 4.
-
In this embodiment the capillary columns 102 are fixed not only
to the holder 4 but also to the holder 6 employing no adhesives.
Therefore, the capillary columns 102 are readily separated from the
holders 4 and 6, and it is also possible to exchange defective capillary
columns.
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Also, as described later, a gel charging apparatus according to the
present invention is provided with a guide rod 130a guiding the holder 4
and fixing means 111 fixing the holder 4, and the holder 4 is provided with
a guide hole 30b through which the guide rod 130a passes and a fixing
part 4b so inclined that thickness of four corners reduce toward an
outward direction to be engaged with the fixing means 111.
-
When clamping the holder plate 6b with respect to the holder
plate 6a with force beyond necessity in the holder 6 on the detection
side, the holder plate 6b is bent due to the combined thickness of the
capillary columns and the rubber plate. In order to prevent this, it is
preferable to provide a groove in the holder 6b in fixing parts of the
holder plates 6a and 6b in response to the thickness of the rubber plate
or to insert a spacer between the holder plates 6a and 6b. It is also
preferable to prepare the holder plate 6b while previously warping The
same opposite to the bent direction. These are effective for preventing
the holder plate 6b from being bent, and effective for maintaining the
force pressing the rubber plate 16 through the clamp 18 and maintaining
holding force around the center of the rubber plate 16.
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For carriage of the capillary cassette, it is convenient to use a
carrier. Fig. 5 shows a capillary cassette comprising a carrier as an
example. (A) is a front elevational view, (B) is a left side elevational view,
and (C) is a top plan view.
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An end of an L-shaped carrier pipe 22 is fixed to a carrier fixture
20 of a holder 4, a holder fixing part 26 is provided on another end of the
carrier pipe 22, and a holder 6 is fixed to the holder fixing part 26 by fixing
screws 28. The holders 4 and 6 are fixed by the carrier pipe 22.
-
In the embodiment of Fig. 5, the carrier 22 is
attachable/detachable, and it is possible to mount the carrier 22 and fix
the holders 4 and 6 when carrying the capillary cassette and detach the
same when mounting the capillary cassette to an electrophoretic
apparatus. However, a capillary cassette integrated with a carrier may
be prepared to be mounted on an electrophoretic apparatus while
comprising the carrier.
-
Fig. 6 shows a schematic block perspective view of one
embodiment of a capillary cassette preparation apparatus, and an
example of a capillary cassette preparation method according to the
present invention is described with reference to this.
-
The embodiment employs quartz glass capillary columns of 300
µm in outer diameter and 100 µm in inner diameter. As a sample
injection side holder, it employs a sample injection side holder 1 holding
capillaries in two dimensions of 16 by 24 of 4.5 mm pitches in coincidence
with a 384-hole microplate used in sample injection.
-
The sample injection side holder 1 is in such a structure that a
supporter consisting of a silicone rubber plate is held between two metal
plates or resin plates in which holes are provided on positions matched
with the 384-hole microplate. A needle 3 (e.g., 0.72 mm in outer
diameter and 0.41 mm in inner diameter) linearly moving in a direction
perpendicular to the plane of the supporter and passing through the
supporter is provided. The needle 3 has an inner diameter larger than
the outer diameter of the capillary columns. A chuck 7 serving as a first
capillary column holder chucking prescribed positions from forward ends
of the capillary columns and inserting the forward ends into the needle 3
is provided, and a guide pipe 5 having an inner diameter larger than the
outer diameter of the capillary columns is provided for guiding the
forward ends of the capillary columns chucked by the chuck 7 into the
needle 3. In order to hold the forward end portions of the capillary
columns made to project from the forward end of the needle 3 in such a
state that the needle 3 passes through the supporter, a chuck 11 serving
as a second capillary column holder is also provided. Furthermore, a
nichrome wire coil 13 preparing detection windows on prescribed
positions from the forward ends of the capillary columns is provided.
The guide pipe 5, the chuck 7 and the nichrome wire coil 13 move on the
same straight line as the needle 3. Furthermore, a cutter 15 cutting the
capillary columns whose single end sides are fixed to the sample injection
side holder 1 in a prescribed length and a capillary feed 17 drawing out
the capillary columns wound on a capillary drum 19 by a constant length
are also provided.
-
It is possible to move the needle 3, the guide pipe 5, the chuck 7
and the nichrome wire coil 13 reciprocally between a position further on
the left side of the position where the needle 3 passes through the
supporter of the sample injection side holder 1 and a position of broken
lines.
-
The capillary cassette preparation method according to the
present invention is formed by the following procedure:
- (1) It mounts the sample injection holder 1 on a non-illustrated
XY stage holding the sample injection side holder 1 and sliding the same
upward, downward, leftward and rightward in the figure while directing the
plane of the supporter of the sample injection holder 1 in a direction
perpendicular to the needle 3, and matches a hole provided on the
sample injection side holder 1 on the moving straight line of the needle 3.
The hole defines a fixing position for the capillary columns.
- (2) In the state where the chuck is on the position of the broken
lines, it extracts a capillary column from the capillary drum 19 using the
capillary feed 17 by the same length as the capillary columns comprised
in the capillary cassette. The extracted capillary column is stored
between the chuck 7 and the capillary feed 17 in a loosened state.
- (3) It passes through the supporter in the sample injection holder
1 by the needle 3.
- (4) It presses the capillary column whose prescribed position
from forward end is chucked by the chuck 7 in the left direction, to make
the forward end pass through the supporter through the guide pipe 5 and
the needle 3. When the needle 3 is clogged with sediment of the
supporter, it pushes out the same with the capillary column. At this
time, the capillary column between the needle 3 and the chuck 7 is
protected by the guide pipe 5, whereby it is possible to prevent the
capillary column from being bent and broken by the force pressing the
sediment of the supporter in the needle 3.
- (5) It chucks the forward end of the capillary column passing
through the needle 3 and projecting on the opposite side of the sample
injection side holder 1 with the chuck 11.
- (6) It removes the coat of the capillary column on the prescribed
position from the forward end of the capillary column with heat of the
nichrome wire coil 13 and prepares a detection window.
- (7) It extracts only the needle 3 from the supporter of the sample
injection side holder 1. The capillary column passing through the
supporter is fixed to the sample injection side holder 1 by elastic force of
the supporter acting in a direction closing the hole.
- (8) In a state chucking and holding the forward end of the
capillary column with the chuck 1, it moves the needle 3, the guide pipe 5,
the chuck 7 and the nichrome wire coil 13 while sliding the same in the
direction of the capillary feed 17 along the capillary column. Thereafter
it cuts the capillary column in a prescribed length from the forward end
with the cutter 15.
- (9) The cut side (detection side) of the capillary column whose
one end is fixed to the sample injection holder 1 is dropped into two slit
holders 21a consisting of two planes having parallel clearances slightly
wider than the outer diameter (300 µm) of the capillary column, and
aligned in cut order, i.e., the order fixed to the sample injection side
holder 1.
- (10) The XY stage moves so that a next fixing position of the
capillary cassette 1 is located on a moving locus of the needle 3.
- (11) It repeats (2) to (10) 384 times, fixes the capillary columns to
all fixing positions of the capillary cassette 1, thereafter aligns end
surfaces of the aligned capillary columns on the detection side with the
slit holders 21a, and mounts/fixes the detection side holder.
-
-
When executing the capillary cassette preparation method
according to the present invention, preparation of a capillary cassette
can be simply and quickly performed.
-
Fig. 7 and Fig. 8 show block diagrams of one embodiment of a
capillary cassette preparation apparatus for more reliably carrying out
the aforementioned capillary cassette preparation method. Fig. 7 is a
plan view and Fig. 8 is a front elevational view. The same reference
numerals are assigned to parts playing the same roles as Fig. 6.
-
Three struts 25a, 25b and 25c are comprised on a substrate 23 in
a direction perpendicular to the plane of the substrate 23. A slide plate
27 comprising a rail 27a sliding a needle unit 3a, a guide unit 5a, a holder
unit 7a and a detection window preparation unit 13a described later in a
direction parallel to the substrate 23 on its side surface is fixed over the
three struts 25a, 25b and 25c. An XY stage is comprised on the
substrate 23 on extension of the longitudinal direction of the slide plate
27 with its moving direction (XY plane) along a vertical direction. A
sample injection side holder 1 having a silicone rubber plate as a
supporter is mounted on the XY stage 29 with the plane of the supporter
perpendicular, and it can be moved upward, downward, leftward and
rightward. A chuck 11 holding and keeping a forward end of a capillary
column is comprised on an opposite side (a side opposite to the slide
plate 27) of the XY stage 29 on extension of the longitudinal direction of
the slide plate 27.
-
A needle 3 having an inner diameter larger than the outer
diameter of the capillary column and passing through the silicone rubber
plate which is the supporter of the sample injection holder 1 is comprised
in the needle unit 3a sliding on the rail 27a comprised on the slide plate
27. A guide pipe 5 having an inner diameter larger than the outer
diameter of the capillary column for making the capillary column between
the needle unit 3a and the holder unit 7a described later not bent is
comprised in the guide unit 5a. A chuck 7 chucking a prescribed
position from the forward end of the capillary column is comprised in the
holder unit 7a. A nichrome wire coil 13 burning and removing a coat of
the capillary column is comprised on the detection window preparation
unit 13a, and the capillary column passes through the center of the
nichrome wire coil 13. A ceramic pipe cutting off transmission of heat
from the nichrome wire coil 13 to the capillary column is provided
between the capillary column and the nichrome wire coil 13 to be
movable along the capillary column. Around the strut 25c on the side
surface of the slide plate 27, a pipe unit 31 guiding the capillary column to
the holder unit 7a is fixed so that the capillary column fed from a capillary
drum 19 described later becomes linear along the slide plate 27.
-
The chuck 11, the needle 3, the guide pipe 5, the chuck 7 and the
nichrome wire coil 13 are provided on a straight line.
-
The capillary drum 19 winding and storing a single long capillary
column is comprised on the substrate 23 on the opposite side to the XY
stage.
-
A cut unit 15a cutting the capillary column after being held by the
sample injection side holder 1 in a prescribed length by moving a cutter
upward and downward is comprised on the strut 25b.
-
A detection side cassette preparation unit 21 is provided on the
substrate 23 between the strut 25a and the strut 25b, and two slit
holders 21a consisting of two planes having parallel clearances slightly
wider than the capillary column outer diameter are comprised on a rotary
plate 21b provided in parallel with the substrate 23. The rotary plate
21b is in contact with the substrate 23 through a fulcrum plate 21c and a
roller 21d, and the longitudinal direction of the rotary plate 21b is fixed in
a direction parallel to the slide plate 27 during an operation of fixing the
capillary column to the sample injection side holder 1, and the clearances
of the slit holders 21a are also directed to the direction parallel to the
slide plate 27, i.e., the direction of the XY stage 29 at this time. It is in
such a structure that the detection side cassette preparation unit 21 can
rotate in the opposite direction to the slide plate 27 by 90° in a direction
parallel to the substrate 23 about a non-illustrated rotation axis provided
in the fulcrum plate 21c in detection side cassette preparation. By
rotating the detection side cassette preparation unit 21 by 90°, the
capillary column enters a state bent by 90° as shown in Fig 1, and is
thereafter fixed to the detection side holder.
-
Fig. 9 shows a flowchart of an operation procedure of the
aforementioned embodiment and description of the operation is made
employing Fig. 7 to Fig. 9.
-
It moves the XY stage and matches a position of the sample
injection side holder 1 for first fixing the capillary column on the straight
line of the
chuck 11, the
needle 3, the guide pipe 5, the
chuck 7 and the
nichrome wire coil 13. The positions of the
needle unit 3a, the
guide
pipe 5a, the
holder unit 7a and the detection
window preparation unit 13a
shown in Fig. 7 and Fig. 8 are initial states. In this state, the forward end
of the capillary column wound on the
capillary drum 19 is fed to the
pipe
unit 31 through a non- illustrated capillary feed having a motor for motive
power. Furthermore, the forward end is fed into the guide pipe 5
through the
nichrome wire coil 13 and the
chuck 7, and the capillary
column is held and kept by the
chuck 7 when the forward end goes out
from the guide pipe 5. While the
nichrome wire coil 13 is fed with a
current and heated, the ceramic pipe intervenes between the
nichrome
wire coil 13 and the capillary column so that the heat of the
nichrome
wire coil 13 is not transmitted to the capillary column.
- (STEP 1) It moves the needle unit 3a and the guide unit 5a in the
left direction with a knob comprised on the guide unit 5a. It moves the
needle unit 3a along the rail 27a leftward until the needle 3 sticks into and
passes through silicon rubber which is the supporter of the sample
injection side holder 1 set on the XY stage 29. At this stage, the holder
unit 7a and the detection window preparation unit 13a follow the guide
unit 5a while keeping a prescribed space and move along the rail 27a.
The capillary column is held by the chuck 7 of the holder unit 7a, to be
pulled in the left direction at the same time.
- (STEP 2) It moves the guide unit 5a further leftward to bring the
same into contact with the needle unit 3a, guides the forward end of the
capillary column going out from the guide pipe 5 into the needle 3, makes
the forward end of the capillary column project from the forward end of
the needle 3, and pushes out sediment of the silicone rubber in the
needle 3.
- (STEP 3) It moves the holder unit 7a further leftward and makes
the forward end of the capillary column further project from the forward
end of the needle 3. At this stage, the ceramic pipe between the
nichrome wire coil 13 of the detection window preparation unit 13a and
the capillary column is moved from the position of the nichrome wire coil
13 on a set time, so that the nichrome wire coil 13 heats a coat on a
prescribed position from the forward end of the capillary column and
starts preparation of a detection window. Since it requires time for
preparation of the detection window, it performs operations of STEP 4
and STEP 5 in parallel.
- (STEP 4) It holds the forward end of the capillary column
projecting from the forward end of the needle 3 with the chuck 11.
- (STEP 5) It moves the needle unit 3a and the guide unit 5a in the
right direction until the needle 3 comes out of the silicone rubber, and
reduces the space between the same and the holder unit 7a. At this
stage, the capillary column is held by the chucks 7 and 11, remains in the
state passing through the silicone rubber, and is fixed to the sample
injection side holder 1 by elastic force of the silicone rubber.
- (STEP 6) It opens the chuck 7, moves the needle unit 3a, the
guide unit 5a, the holder unit 7a and the detection window preparation
unit 13a rightward, and returns the holder unit 7a and the detection
window preparation unit 13a to the positions of the initial states. At this
stage, the space between the guide unit 5a and the holder unit 7a is
shorter than the initial state.
- (STEP 7) The holder unit 7a closes the chuck 7 on the position in
the initial state, and holds the capillary column.
- (STEP 8) It moves the cut unit 15 upward, and cuts the capillary
column with the comprised cutter. The cut portion defines the forward
end of a next capillary column. An end of the capillary column cut in the
prescribed length is fixed to the sample injection side holder 1, and
another end is dropped in the clearance of the slit holders 21a. The
capillary column fixed to the sample injection side holder 1 is
successively dropped in the clearance of the slit holders 21a, and
planarly arranged in a line.
- (STEP 9) It moves the needle unit 3a and the guide unit 5a
leftward and returns the space between the guide unit 5a and the holder
unit 7a to the position in the initial state. Thus, the needle unit 3a, the
guide unit 5a, the holder unit 7a and the detection window preparation
unit 13a return to the initial positions.
- (STEP 10) It opens the chuck 11 and releases the chucked
forward end of the capillary column. Thereafter it moves the XY stage
and then matches the position of the sample injection side holder 1 for
fixing the capillary column on the straight line of the chuck 11, the needle
3, the guide pipe 5, the chuck 7 and the nichrome wire coil 13. When
fixation of all capillary columns ends, it advances to STEP 11, otherwise
returns to STEP 1.
- (STEP 11) It moves the XY stage 29 to the lowermost position
for preparing a capillary cassette, and moves the sample injection side
holder 1 to a prescribed position.
- (STEP 12) In such a state that 384 capillary columns are
arranged in a line on a plane in the two slit holders 21a, it rotates the
rotary plate 21b of the detection side cassette preparation unit 21 in the
opposite direction to the slide plate 27 with a roller 4d by 90° about a
non-illustrated rotation axis provided in the fulcrum plate 21c. At this
time, each capillary column is bent by 90°.
- (STEP 13) It rectilinearly aligns end surfaces of the 384 capillary
columns going out from the slit holders 21a with a prescribed jig (angle).
At this time, a detection window provided in each capillary column is
positioned between the two slit holders 21a.
- (STEP 14) It holds and fixes the 384 capillary columns with
clamps comprised in the slit holders 21a holding the capillary columns
positioned in the clearances of the slit holders 21a.
- (STEP 15) It matches the detection side cassette along between
the two slit holders 21a and screws the same.
-
-
By operating a lever in accordance with the aforementioned
procedure, capillary columns of a predetermined length are aligned in set
order, so that a capillary cassette can be prepared.
-
While a manual example has been shown as to the operation in
the embodiment, it is preferable to make automatization with a driving
source such as a motor or an air cylinder. Thus, preparation of the
capillary cassette can be further simply performed without failure.
-
While the capillary columns have been charged one by one in the
embodiment, it is preferable to arrange a plurality of for example, 16
needles, and capillary columns and simultaneously move the same.
Thus, preparation of the capillary cassette in a shorter time can be
performed.
-
Another embodiment forming a detection window on the position
of a detected
portion 2c in a capillary cassette 2 is described with
reference to Fig. 10.
- (A) After fixing sample injection sides of 384 capillary columns
102 not subjected to coat removal with a holder, it aligns other end sides,
i.e., detection sides in a line planary of a flat plate 204 in a close contact
manner. A slit longer than the arrangement width of the capillary
columns 102 is opened in the flat plate 204 in order to prevent the
capillary columns 102 from adhering to the flat plate 204 by melting of
coats through a coat removal operation, and it performs positioning so
that a detection window formation planned region 102a comes onto the
slit
- (B) It further overlaps another flat plate 206 on the capillary
columns 102. A slot longer than the arrangement width of the capillary
columns 102 is opened also in the flat plate 206, and it performs
positioning so that the slot comes to the detection window formation
planned region 102. Thus, it holds and fixes the capillary columns 102
between the two flat plates 204 and 206.
- (C) It feeds a current to a nichrome wire coil 208 to burn the
coats and makes it heat red. The nichrome wire coil 208 is that winding
a 40 cm long of a nichrome wire of 0.5 mm in diameter, wound into the
form of a coil of about 3 mm in diameter and 2cm in length. The
energization quantity is, for example, 5 A.
It brings the red-heating nichrome wire coil 208 into contact with
the capillary columns 102 while directing the same to an end portion of
the detection window formation planned region 102a through the slit of
the flat plate 206 so that the longitudinal direction of the nichrome wire
coil 108 is along the arrangement direction (the direction perpendicular to
the longitudinal direction of the capillary columns 102) of the capillary
columns 102.
- (D) It brings the nichrome wire coil 208 into contact with the
capillary columns 102 for about 10 seconds, burns and removes the
coats on the positions, and thereafter moves the nichrome wire coil 208
along the slot of the flat plate 208 toward the other end portion direction
of the detection window formation planned region 102a while partially
overlapping portions for bringing the nichrome wire coil 208 into contact
so that the coats do not remain on the detection window formation
planned region 102a. At this time, coats on surfaces opposite to the
surfaces with which the nichrome wire coil 208 comes into contact are
also burned and removed.
-
-
When the coats of the respective capillary columns 102 melted in
positions separate from the nichrome wire coil 208 in burning are cooled
and solidified, the adjacent capillary columns 102 are fused to each other
by the coats once melted. Thus, the overall plurality capillary columns
102 arranged in a line form a flat cable.
-
While the embodiment has used the coiled nichrome wire as a
means of heating a long and narrow ceramic heater or the like can also
be used as the heating means. When the number of the capillary
columns fixed to the holder is large, the width when planarly aligning the
capillary columns in a line becomes large. When lengthening the
nichrome wire coil for reducing the time required for removal of the coats,
its strength weakens in such a manner that the nichrome wire coil is bent
and deformed while preparing a detection window by pressing the same
against the capillary columns and cannot indeed come into contact with
the planarly aligned capillary columns, and it may become necessary to
repeatedly perform the burning operation over and over. When using a
ceramic heater longer than the width when planarly aligning the capillary
columns in a line in such a case, it is possible to form a detection window
at once and hence the detection window forming time can be reduced.
-
Fig. 11, Fig. 12 and Fig. 13 show schematic block diagrams of one
embodiment of a capillary gel charging apparatus according to the
present invention. Fig. 11 is a front elevational view showing a closing
means part and a fixing means part in a partially fragmented manner, Fig.
12 is a plan view showing an upper portion of a pump means storage box
and a timer unit set therein in a partially fragmented manner, and Fig. 13
is a sectional view showing the pump means storage box in a partially
fragmented manner, showing the same while cutting the same along an
A-A line position of Fig. 12.
-
A chamber 103 in which the sample injection side holder 4 shown
in Fig. 2, Fig. 3 and Fig. 4 is fixed to form a closed space, a pump means
storage box 105 comprising pump means pressurizing or decompressing
the chamber 103 therein, detection side holder fixing means 107 and
detection side gel vessel fixing means 129 are comprised on a substrate
101.
-
The chamber 103 is formed by 170 mm × 170mm square acrylic
plates 103a and 103c of 10 mm in thickness for closing an upper portion
and a bottom portion, and an acrylic pipe 103b of 165 mm in outer shape
and 145 mm in inner diameter for closing side portions. Clearances
between both sections of the acrylic pipe 103b and the acrylic plates
103a and 103c are sealed through a silicone rubber packing. An
opening 113 of a rectangular hole of 80 mm by 115 mm is provided on the
acrylic plate 103, and the sample injection side holder 4 is mounted
thereon. A groove is dug around the rectangular hole opening 113, and
an annular silicone sponge 113a having a circular section is fit into the
groove. About half the silicone sponge 113a goes out from the surface
of the acrylic plate 103a, to seal a clearance between the acrylic plate
103a and the sample injection side holder 4. Four clamps 111 for fixing
the sample injection side holder 4 to the acrylic plate 103a are comprised
on the acrylic plate 103a and engage with fixing parts 4d on four corners
of the sample injection holder 4, so that the holder 4 is pressed against
the sponge 113a and closes the space of the chamber 103 by tightening
the clamps 111.
-
Two joints are provided on a side surface (cylindrical surface) of
the acrylic pipe 103, and a pump 119 pressurizing or decompressing the
chamber 103 is connected to one joint 115. An electromagnetic valve
121 releasing the pressure in the pressurized or decompressed chamber
103 to the atmospheric pressure through a needle valve 117 is
connected to the other joint and its return pressure speed is adjusted by
the needle valve 117.
-
In the pump means storage box 105, a timer 123a controlling a
working time of the pump 119 and a timer 123b controlling a working time
of the electromagnetic valve 121 are stored in addition to the pump 119
and the electromagnetic valve 121.
-
A detection side gel vessel 129 storing a gel solution when
inhaling the gel solution from the detection side is comprised in the
detection side gel vessel fixing means 129. It uses a polyacrylamide
solution of 5 %T and 5 %C, for example, for the gel solution.
-
The operation shall now be described. The embodiment has
employed the capillary cassette comprising the 384 capillary columns
102 shown in Fig. 2, Fig. 3 and Fig. 4.
-
Sample injection sides of the 384 capillary columns 102 are
arranged in two dimensions of 16 by 24 of 4.5 mm pitches in coincidence
with a commercially available microplate by the sample injection holder 4
and fixed with excellent airtightness, and detection sides thereof are
planarly closely fixed in a line by the detection side holder 6.
-
When performing gel charging by decompressing the chamber 3, it
stores the gel solution in a detection side gel vessel 129a. It fixes the
fixing parts 4d on the four corners of the detection side holder 4 by the
four clamps 111 provided on the acrylic plate 103a so that the forward
ends on the detection sides of the capillary columns 102 are dipped into
the gel solution stored in the detection side gel vessel 129a, presses the
sample injection side holder 4 against the silicone sponge 113a by
tightening the clamps 111, and closely fixes the same to the acrylic plate
103a. It decompresses the chamber 103 with the pump 119 for
decompressing the inner parts of the capillary columns 102, and inhales
the gel solution from the forward ends on the detection sides. The gel
solution is charged into the capillary columns 102, and it is preferable to
comprise a non-illustrated vessel in the chamber 103 to receive an
excess amount of gel solution overflowing from the sample injection side.
-
When performing gel charging by pressurizing the inner part of the
chamber 103, it comprises a non-illustrated vessel storing the gel
solution and vacates the vessel 129a. It adjusts the length of the
capillary columns projecting from the holder 4 and the height of the
vessel storing the gel vessel so that the forward ends of all capillary
columns on the sample injection side are dipped into the gel vessel when
fixing the fixing parts 4d on the four corners of the sample injection side
holder 4 with the four clamps 111 provided on the acrylic plate 103a.
It fixes the detection side holder 6 to the detection side holder fixing
means 107. In this case, the sample injection side holder 4 is pressed
against the silicone sponge 113 by tightening the clamps 111 and closely
fixed to the acrylic plate 103a, so that the space inside the chamber 103
enclosed with the sample injection side holder 4, the acrylic plates 103a
and 103b and the acrylic pipe 103 is closed. It pressurizes the chamber
103 with the pump 119 and pushes the gel solution into the capillary
columns by the pressure. The gel solution is charged into the capillary
columns 102, and it receives an excess amount of gel solution
overflowing from the detection side by the detection side gel vessel 129a.
-
Both in the case of performing decompression and, in the case of
performing pressurization, it times the time when the gel solution is
charged into all capillary columns 102 and previously sets the working
times of the pump 119 and the electromagnetic valve 121 with the timers
123a and 123b. When charging of the gel solution is started by a RUN
button, the pump 119 starts working and the chamber 103 is
decompressed or pressurized. Continuing the decompression or the
pressurization by the time set at the timer 123a, the pump 119 stops at a
prescribed time. The inner part of the chamber 103 is still
decompressed or pressurized even if the pump 119 stops, whereby the
electromagnetic valve 121 opens when the time set at the timer 123b
elapses so that the pressure in the chamber 103 is released to the
atmosphere through the needle valve 117. At this time, the speed at
which the internal pressure of the chamber 103 returns is adjusted by
the needle valve 117.
-
Decompression·pressurization in the chamber 103 can be
selected depending on to which one of an exhaust port and a suction
port of the pump 119 a pipe connected to the joint 115 is connected.
-
In order to prevent air from being mixed into the gel charged into
the capillary columns since the degree of decompression or
pressurization of the chamber 103 is large, it is preferable to control the
degree of decompression or pressurization. This control can be
performed by a throttle comprised in the needle valve 117 while opening
the electromagnetic valve 121 also when the pump 119 operates by the
timer 123a. In this case, the electromagnetic valve 121 temporarily
closes when the set time of the timer 123a elapses, and opens again
after a lapse of the time set in the timer 123b to release the chamber 103
to the atmosphere.
-
While it is of a pump storing type in the embodiment it is
preferable to render a pressure generation source outside and connect
the same to, for example, a high pressure cylinder or a vacuum pump,
when the charged gel solution is that having high viscosity such as a flow
gel, and it is necessary to generate a high pressure. When employing a
high pressure cylinder, it is preferable to provide a new electromagnetic
valve, control the same with a timer, and introduce compressed gas in
the cylinder into the chamber.
-
While the embodiment has used a diaphragm pump for enabling
switching of decompression·exhaust, a pump of only suction or only
exhaust may be employed.
Industrial Availability
-
As described above, the capillary cassette according to the
present invention is suitable for use as a migration part of a multi-capillary
electrophoretic apparatus used for separating/analyzing nucleic
acid, protein, peptide, sugar or the like, and particularly suitable for
employment for analysis of the base sequence of DNA.